Title:	Thermostable xylanases
Document Type and Number:	United States Patent 7060482
Link to this Page:	http://www.freepatentsonline.com/7060482.html
Abstract:	The present invention is directed to thermostable xylanase enzymes are suitable for feed pelleting applications. The novel xylanase enzymes comprise at least 40% of their optimal activity from a pH range from about pH 3.5 to about pH 6.0, and from about 40 to about 60.degree. C., and exhibit at least 30% of their optimal activity after a pre-incubation step for 30 minutes at 70.degree. C. in the presence of 40% glycerol. Also disclosed are modified xylanase molecules comprising either a basic amino acid at position 162 (TrX numbering), or its equivalent position in other xylanase molecules, at least one disulfide bridge, or a combination thereof. The thermostable xylanase molecules of the present invention have a physiological temperature and pH optima and are useful as animal feeds additives since they can withstand the heat associated with feed sterilization and pellet formation, yet they exhibit optimal activity within an animal to aid in breakdown of ingested feed.
Inventors:	Sung, Wing L.; Tolan, Jeffrey S.;
Application Number:	856025
Filing Date:	1999-11-16
Publication Date:	2006-06-13
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Assignee:	National Research Council of Canada (Ottawa, CA)
Current Classes:	435 / 200 , 426 / 531, 426 / 56, 435 / 183, 435 / 210, 435 / 4, 435 / 6, 435 / 69.1, 530 / 350, 536 / 23.2, 536 / 23.4, 536 / 23.7
International Classes:	C12N 9/00 (20060101); A23L 1/31 (20060101); C07H 21/04 (20060101); C12P 21/06 (20060101)
Field of Search:	536/23.2,23.7,23.24,23.4 435/252.3,254.1,254.11,254.6,4,6,15,69.1,183,195,200,320.1 530/350 426/56,531
US Patent References:	5405769 April 1995 Campbell et al. 5759840 June 1998 Sung et al.
Foreign Patent References:	0828002 Mar., 1998 EP WO 9424270 Oct., 1994 WO WO 9529997 Nov., 1995 WO
Other References:	Wakarchuk et al. "Thermostabilization of the Bacillus Circulans Xylanase by the Introduction of Disulfide Bonds," Protein Engineering 7(11):1379-1386 (1994). cited by other . Marquardt et al. "Recommendations for future research on the use of enzymes in animal feeds," Enzymes in Poultry and Swine Nutrition, pp. 129-138 (Jun. 1997). cited by other . Liu et al. "Exogenous enzymes for pig diets: an overview," Enzymes in Poultry and Swine Nutrition, pp. 115-128 (Jun. 1997). cited by other . Guenter "Practical experience with the use of enzymes," Enzymes in Poultry and Swine Nutrition, pp. 53-62. cited by other . Arase et al. "Stabilization of xylanase by random mutagenesis," Federation of European Biochemical Societies, 316(2):123-127 (Jan. 1993). cited by other . Cowan "Protein stability at high temperatures," Essays in Biochemistry, vol. 29, pp. 193-207 (1995). cited by other . Gupta "Thermostabilization of Proteins," Biotechnology and Applied Biochemistry, vol. 14, pp. 1-11 (1991). cited by other . Paloheimo et al. "Thermostable Xylanases Produced by Recombinant Trichoderma Reesei for Pulp Bleaching," Carbohydrases from Trichoderma reesei and Other Microorganisms, pp. 255-264 (1997). cited by other . Sung et al. "Overexpression of the Bacillus subtilis and circulans Xylanases in Escherichia coili ," Protein Expression and Purification, vol. 4, pp. 200-206 (1993). cited by other . Sung et al. "Short synthetic oligodeoxyribonucleotide leader sequences enhance accumulation of human proinsulin synthesized in Escherichia coli, " Proc. Natl. Acad. Sci. USA, vol. 83, pp. 561-565. cited by other . Vivcros et al. "Effect of enzyme supplementation of a diet based on barley, and autoclave treatment, on apparent digestibility, growth performance and gut morphology of broilers," Animal Feed Science and Technology, vol. 48, pp. 237-251 (1994). cited by other.
Primary Examiner:	Rao; Manjunath N.
Attorney, Agent or Firm:	Needle & Rosenberg PC
Parent Case Data:	This application is a claims priority to U.S. Provisional Application No. 60/108,504, filed Nov. 16, 1998. The present invention relates to thermostable xylanase enzymes. More specifically, the present invention is directed to thermostable xylanase enzymes that exhibit high activity at or near physiological pH and temperature, and their use in feed pelleting applications.

Claims:

What is claimed is: 1. An isolated, modified, Family 11 xylanase comprising at least one intramolecular disulfide bond and a basic amino acid at position 162, said position determined from sequence alignment of said modified xylanase with Trichoderma reesei xylanase II amino acid sequence defined in SEQ ID NO: 16, said modified xylanase exhibiting at least 40% of optimal activity from about pH 3.5 to about pH 6.0, and from about 40 to about 60.degree. C., said modified xylanase exhibiting increased thermostablity relative to the corresponding natural xylanase. 2. The modified xylanase of claim 1 selected from the group consisting of TrX-162H-DS1 (SEQ ID NO: 62), TrX-162H-DS2 (SEQ ID NO: 63), TrX-162H-DS4 (SEQ ID NO: 64) and TrX-DS8 (SEQ ID NO: 65). 3. The modified xylanase of claim 2 wherein said xylanase is TrX-162H-DS1 (SEQ ID NO: 62). 4. The modified xylanase of claim 2, wherein said xylanase is TrX-162H-DS2 (SEQ ID NO: 63). 5. The modified xylanase of claim 2, wherein said xylanase is TrX-162H-DS4 (SEQ ID NO: 64). 6. The modified xylanase of claim 2, wherein said xylanase is TrX-DS8 (SEQ ID NO: 65). 7. A method of preparing animal feed comprising applying the isolated xylanase of claim 1 onto said animal feed to produce a xylanase-animal feed combination, and heat sterilizing said xylanase-animal feed combination. 8. The method of claim 7, wherein said animal feed is a poultry or swine feed. 9. The modified xylanase of claim 1, wherein the basic amino acid is selected from the group consisting of lysine, arginine, and histidine.

Description: